Primary Site Of Action Research Articles

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.

Exploring the Link Between Ativyayama (Overexercise) and Srotodusti, with Emphasis on Pranavaha Srotas: A Comprehensive Scientific Analysis

Vyayama, a specialized concept in Ayurveda, serves as both the cause and remedy for various illnesses. While physical exercise is beneficial, excessive engagement in it can lead to negative health outcomes. Over-exertion aggravates the Vata Dosha, which in turn affects the qualities of the channels (Srotas), making them Ruksha and Khara. Consequently, Vyayama becomes a primary factor in the disturbance of the channels Pranavaha, Asthivaha, and Swedavaha Srotas. Conversely, insufficient exercise disrupts the Medovaha Srotas. The Pranavaha Srotas serves as the vital channel responsible for carrying Vata, especially Prana, which sustains the vitality of every cell. Disruption in the structure or function of this channel leads to illness. When exercise is excessive, it can lead to Hridayoparodha resulting in impairment of the primary site of action. This can manifest as acute conditions (as myocardial infarction) or chronic conditions (as Shosha). It's important to engage in exercise appropriately according to Bala and Ritu to preserve health. This article explores the scientific evidence supporting the notion that excessive exercise (Ativyayama) can disturb the balance of the body's channels, particularly the Pranavaha Srotas. It offers a thorough examination of the relationship between overexercise and channel disturbances, providing insights into potential risks and preventive measures.

Preclinical pharmacokinetics, pharmacodynamics, and toxicity of novel small-molecule GPR119 agonists to treat type-2 diabetes and obesity

The escalating global prevalence of type-2 diabetes (T2D) and obesity necessitates the development of novel oral medications. Agonism at G-protein coupled receptor-119 (GPR119) has been recognized for modulation of metabolic homeostasis in T2D, obesity, and fatty liver disease. However, off-target effects have impeded the advancement of synthetic GPR119 agonist drug candidates. Non-systemic, gut-restricted GPR119 agonism is suggested as an alternative strategy that may locally stimulate intestinal enteroendocrine cells (EEC) for incretin secretion, without the need for systemic drug availability, consequently alleviating conventional class-related side effects. Herein, we report the preclinical acute safety, efficacy, and pharmacokinetics (PK) of novel GPR119 agonist compounds ps297 and ps318 that potentially target gut EEC for incretin secretion. In a proof-of-efficacy study, both compounds demonstrated glucagon-like peptide-1 (GLP-1) secretion capability during glucose and mixed-meal tolerance tests in healthy mice. Furthermore, co-administration of sitagliptin with investigational compounds in diabetic db/db mice resulted in synergism, with GLP-1 concentrations rising by three-fold. Both ps297 and ps318 exhibited low gut permeability assessed in the in-vitro Caco-2 cell model. A single oral dose PK study conducted on healthy mice demonstrated poor systemic bioavailability of both agents. PK measures (mean ± SD) for compound ps297 (Cmax 23 ± 19 ng/mL, Tmax range 0.5 – 1 h, AUC0–24 h 19.6 ± 21 h*ng/mL) and ps318 (Cmax 75 ± 22 ng/mL, Tmax range 0.25 – 0.5 h, AUC0–24 h 35 ± 23 h*ng/mL) suggest poor oral absorption. Additionally, examinations of drug excretion patterns in mice revealed that around 25 % (ps297) and 4 % (ps318) of the drugs were excreted through faeces as an unchanged form, while negligible drug concentrations (<0.005 %) were excreted in the urine. These acute PK/PD assessments suggest the gut is a primary site of action for both agents. Toxicity assessments conducted in the zebrafish and healthy mice models confirmed the safety and tolerability of both compounds. Future chronic in-vivo studies in relevant disease models will be essential to confirm the long-term safety and efficacy of these novel compounds.

The processing intermediate of human amylin, pro-amylin(1–48), has in vivo and in vitro bioactivity

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin’s primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

Improving human sperm motility via red and near-infrared laser irradiation: in-vitro study

Improved sperm motility is necessary for successful sperm passage through the female genital system, efficacious fertilization, and a greater probability of pregnancy. By stimulating the mitochondrial respiratory chain, low-level laser photobiomodulation has been shown to increase sperm motility and velocity. The respiratory chain in mitochondria is the primary site of action for cytochrome c oxidase because it can absorb light in the visible and infrared ranges. The present study aimed to investigate the effects of red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both on human spermatozoa motility and DNA integrity at different doses. An in-vitro controlled trial was performed in Al Zahraa university hospital laboratory using thirty fresh human semen specimens. Samples were exposed to red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both for various irradiation times. Sperm motility for the test and control aliquots was assessed as recommended in the manual of WHO-2021. Sperm chromatin integrity was evaluated using the Sperm Chromatin Structure Assay. Results revealed almost 70%, 80% and 100% increase in the total motility after 3 min of the 650-nm, 980-nm and the combined laser irradiation, respectively. Additionally, the Sperm Chromatin Dispersion assay was carried out on sperm heads utilizing human sperm DNA fragmentation, demonstrating that none of the three laser types had any discernible effects.Graphical abstract

The muscle-enriched myokine Musclin impairs beige fat thermogenesis and systemic energy homeostasis via Tfr1/PKA signaling in male mice

Skeletal muscle and thermogenic adipose tissue are both critical for the maintenance of body temperature in mammals. However, whether these two tissues are interconnected to modulate thermogenesis and metabolic homeostasis in response to thermal stress remains inconclusive. Here, we report that human and mouse obesity is associated with elevated Musclin levels in both muscle and circulation. Intriguingly, muscle expression of Musclin is markedly increased or decreased when the male mice are housed in thermoneutral or chronic cool conditions, respectively. Beige fat is then identified as the primary site of Musclin action. Muscle-transgenic or AAV-mediated overexpression of Musclin attenuates beige fat thermogenesis, thereby exacerbating diet-induced obesity and metabolic disorders in male mice. Conversely, Musclin inactivation by muscle-specific ablation or neutralizing antibody treatment promotes beige fat thermogenesis and improves metabolic homeostasis in male mice. Mechanistically, Musclin binds to transferrin receptor 1 (Tfr1) and antagonizes Tfr1-mediated cAMP/PKA-dependent thermogenic induction in beige adipocytes. This work defines the temperature-sensitive myokine Musclin as a negative regulator of adipose thermogenesis that exacerbates the deterioration of metabolic health in obese male mice and thus provides a framework for the therapeutic targeting of this endocrine pathway.

Homo medialiteratus and the media literacy proxy war: mapping the U.S. response to digital dismisinfo

ABSTRACT Alongside climate change, democracy, and public health, digital disinformation and misinformation, or digital dismisinfo, stands among the most pressing of global challenges. But how are societies responding to that challenge? Who are the key actors, what solutions do they propose or enact, and how does their work interact with that of others? To address these questions, the authors undertook a visual network analysis of the U.S.-based response to digital dismisinfo. Tracing the connections and disconnections across networked actors (e.g., platforms, nonprofits, governance, etc.) rendered visible a growing convergence towards the media-consuming individual as the primary site of action and correction with respect to digital dismisinfo. The authors argue that through such networked dynamics emerges the figure of homo medialiteratus, the media-consuming individual who must bootstrap their way to truth in the face of an unrelenting tide of digital dismisinfo.

Pasteurized Akkermansia muciniphila improves glucose metabolism is linked with increased hypothalamic nitric oxide release

Background and objectivePasteurized Akkermansia muciniphila cells have shown anti-diabetic effects in rodents and human. Although, its primary site of action consists in maintaining the gut barrier function, there are no study exploring if A. muciniphila controls glycemia via a gut to brain axis. Targeting the gut motility represents an alternative pathway to treat hyperglycemia. Here, we tested the impact of pasteurized A. muciniphila on gut motility, gut-brain axis and glucose metabolism. MethodsWe used mice fed a 45% high-fat (HFD) treated or not with pasteurized A. muciniphila MucT during 12 weeks. We measured the effects of the treatment on body weight gain, glucose metabolism (insulin, glycemia, glucose tolerance), gut contraction and enteric neurotransmitter release, and hypothalamic nitric oxide (NO) release. ResultsWe show that pasteurized A. muciniphila exerts positive effects on different metabolic parameters such as body weight, fat mass, insulin, glycemia and glucose tolerance. This could be explained by the ability of pasteurized A. muciniphila supplementation to decrease duodenal contraction and to increase hypothalamic NO release in HFD mice. ConclusionWe demonstrate a novel mode of action of pasteurized A. muciniphila explaining its beneficial impact on the control of glycemia in a preclinical model of type 2 diabetes via gut-brain axis signaling.

Case Report: Diabetic ketoacidosis after co-administration of empagliflozin and probenecid

Sodium-glucose cotransporter-2 (SGLT2) inhibitors are filtered and secreted to their primary site of action in the proximal tubule of the kidney. Many commonly used medications have potential to diminish renal elimination of SGLT2 inhibitors by inhibiting their tubular secretion. We present a case of severe diabetic ketoacidosis (DKA) in a patient with type 2 diabetes occurring several days after co-prescription of empagliflozin and probenecid. Other than the recent introduction of empagliflozin, no cause for the DKA episode was apparent. A pharmacokinetic interaction between probenecid and empagliflozin, involving organic anion transporter 3 (OAT3), reduces proximal tubular secretion of empagliflozin. Co-administration of an OAT3 inhibitor, such as probenecid, with empagliflozin may increase patient exposure and diminish glucosuric response to the SGLT2 inhibitor, thereby increasing the risk of DKA. We suggest that clinicians exercise caution when prescribing empagliflozin alongside inhibitors of OAT3 in patients with type 2 diabetes.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Radium– 223 Dichloride Related Toxicity in Post – Chemotherapy Castrate Resistant Prostate Cancer Patients Treated for Bone Metastases

Background: Radiation pharmacokinetics and pharmacodynamics of Radium point to bone as its site of primary uptake and action. It is therefore very important to investigate the hematological and other toxicity of the radiopharmaceutical Radium -223 dichloride in the wake of its introduction for treatment of bone metastases. Methods: Five Patients with post-chemotherapy castrate resistant prostate cancer with bone metastases received 50kBq/ kg body weight of Radium-223 Dichloride injection every 4 weeks for 5 cycles. 1. Screening 2. Treatment (including an End of Treatment Visit) 3. Follow-up Results: The ranges of measured parameters for 4 patients at the end of 5th cycle (20th week) were within normal limits: Hb : Wk 20 = 110 -126g/l, pre-treatment = 110-136g/l (normal range 120-170) Wbc: Wk 20 = 3.3-6.2x109/l, Pre-treatment=3.5-8.9x109/l (normal range 4.5-10) Platelets: Wk 20 = 156-241x109/l, pre-treatment = 206-302x109/l (normal range 150-400). Alkaline Phosphatase: Wk 20 = 43-128IU/l, Pre-treatment = 57-253IU/l (normal Range 40-120). The 5th Patient developed bone marrow failure in the 12th week, with concurrent flaring of his alkaline phosphatase value. Conclusion: The results of this study suggest that Radium-223 dichloride at dose of 50kbq/kg is safe for palliative treatment of bone pains in metastatic bone diseases. The 5th patient’s bone marrow failure cause may be attributed to several factors such as 1. Direct effect of the drug on bone tissues 2. Drug induced allergic response 3. myelosuppression worsened by concomitant radiotherapy

Metformin acts in the gut and induces gut-liver crosstalk

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Community-based participatory climate action

Non-technical summary Improving the flow of information between governments and local communities is paramount to achieving effective climate change mitigation and adaptation. We propose five pathways to deepen participation and improve community-based climate action. The pathways can be summarized as visualization, simulations to practice decision-making, participatory budgeting and planning, environmental civic service, and education and curriculum development. These pathways contribute to improving governance by consolidating in governments the practice of soliciting and incorporating community participation while simultaneously giving communities the tools and knowledge needed to become active contributors to climate change adaptation and mitigation measures. Technical summary Community participation is considered a key component in the design of responses to climate change. Substantial engagement of local communities is required to ensure information flow between governments and communities, but also because local communities are the primary sites of adaptation action. However, frontline communities are often excluded from decision-making and implementation processes due to political choices or failures to identify ways to make participatory frameworks more inclusive. Climate action requires the active engagement of communities in making consequential decisions, or what we term deepened participation. We propose five pathways to deepen participation: visualization, simulations to practice decision-making, participatory budgeting and planning, environmental civic service, and education and curriculum development. The five pathways identify strategies that can be incorporated into existing organizational and institutional frameworks or used to create new ones. Shortcomings related to each strategy are identified. Reflection by communities and governments is encouraged as they choose which participatory technique(s) to adopt. Social media summary Climate action requires the active engagement of communities. Learn five pathways to get started deepening participation.

Kidney-Protective Effects of SGLT2 Inhibitors.

The sodium-glucose cotransporter 2 (SGLT2) inhibitors have become an integral part of clinical practice guidelines to slow the progression of CKD in patients with and without diabetes mellitus. Although initially developed as antihyperglycemic drugs, their effect on the kidney is multifactorial resulting from profuse glycosuria and natriuresis consequent to their primary site of action. Hemodynamic and metabolic changes ensue that mediate kidney-protective effects, including ( 1 ) decreased workload of proximal tubular cells and prevention of aberrant increases in glycolysis, contributing to a decreased risk of AKI; ( 2 ) lowering of intraglomerular pressure by activating tubular glomerular feedback and reductions in BP and tissue sodium content; ( 3 ) initiation of nutrient-sensing pathways reminiscent of starvation activating ketogenesis, increased autophagy, and restoration of carbon flow through the mitochondria without production of reactive oxygen species; ( 4 ) body weight loss without a reduction in basal metabolic rate due to increases in nonshivering thermogenesis; and ( 5 ) favorable changes in quantity and characteristics of perirenal fat leading to decreased release of adipokines, which adversely affect the glomerular capillary and signal increased sympathetic outflow. Additionally, these drugs stimulate phosphate and magnesium reabsorption and increase uric acid excretion. Familiarity with kidney-specific mechanisms of action, potential changes in kidney function, and/or alterations in electrolytes and volume status, which are induced by these widely prescribed drugs, will facilitate usage in the patients for whom they are indicated.

Polycomb EZH1 regulates cell cycle/5-fluorouracil sensitivity of neuroblastoma cells in concert with MYCN.

In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

Does the age of packed red blood cells, donor sex or sex mismatch affect the sublingual microcirculation in critically ill intensive care unit patients? A secondary interpretation of a retrospective analysis

In vitro studies have thoroughly documented age-dependent impact of storage lesions in packed red blood cells (pRBC) on erythrocyte oxygen carrying capacity. While studies have examined the effect of pRBC age on patient outcome only few data exist on the microcirculation as their primary site of action. In this secondary analysis we examined the relationship between age of pRBC and changes of microcirculatory flow (MCF) in 54 patients based on data from the Basel Bedside assessment Microcirculation Transfusion Limit study (Ba2MiTraL) on effects of pRBC on sublingual MCF. Mean change from pre- to post-transfusion proportion of perfused vessels (∆PPV) was + 8.8% (IQR − 0.5 to 22.5), 5.5% (IQR 0.1 to 10.1), and + 4.7% (IQR − 2.1 to 6.5) after transfusion of fresh (≤ 14 days old), medium (15 to 34 days old), and old (≥ 35 days old) pRBC, respectively. Values for the microcirculatory flow index (MFI) were + 0.22 (IQR − 0.1 to 0.6), + 0.22 (IQR 0.0 to 0.3), and + 0.06 (IQR − 0.1 to 0.3) for the fresh, medium, and old pRBC age groups, respectively. Lower ∆PPV and transfusion of older blood correlated with a higher Sequential Organ Failure Assessment (SOFA) score of patients upon admission to the intensive care unit (ICU) (p = 0.01). However, regression models showed no overall significant correlation between pRBC age and ∆PPV (p = 0.2). Donor or recipient sex had no influence. We detected no significant effect of pRBC on microcirculation. Patients with a higher SOFA score upon ICU admission might experience a negative effect on the ∆PPV after transfusion of older blood.

Proteasomal turnover of the RhoGAP tumor suppressor DLC1 is regulated by HECTD1 and USP7

The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin–proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.

Biochemical Action of Anesthesia Revealed

Anesthesia, the term was coined by Oliver Wendell Holmes in 1846, to portray insensibility to pain induced by some drug, shortly after the demonstration of loss of consciousness by inhaled ether during a surgical procedure. General anesthesia, for almost two centuries (175 years), has made scientists wonder that how under its influence a person loses consciousness and his body becomes insensitive to pain yet the body continues performing normal vital physiologic functions. This clearly demonstrates that the central nervous system (CNS) is their primary site of action, where the nerve transmission is reduced at the synapses which release the neurotransmitters for action in the body. The only information researchers had was that general anesthesia works by suspending signals from the brain and body and blocks any record of pain or event during this period.

Choroid plexus-selective inactivation of adenosine A2A receptors protects against T cell infiltration and experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A2AR-mediated protection remains undetermined.MethodsIn the EAE model, we assessed A2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8–12 or 8–14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A2AR via intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration.ResultsWe found the specific upregulation of A2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8–12 or 8–14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A2AR knock-down attenuated the pathogenic infiltration of Th17+ cells across the CP via inhibiting the CCR6–CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in the cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration.ConclusionThese findings define the CP niche as one of the primary sites of A2AR action, whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

Differential effects of single fatty acids and fatty acid mixtures on the phosphoinositide 3-kinase/Akt/eNOS pathway in endothelial cells

ScopeDietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways.MethodsTo examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting.ResultsOleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110β) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone.ConclusionOur findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.